A physical map of the chromosome of Neisseria gonorrhoeae FA1090 has been constructed. Digestion of strain FA1090 DNA with NheI, SpeI, BgM, or Pacl resulted in a limited number of fragments that were resolved by contour-clamped homogeneous electric field electrophoresis. The estimated genome size was 2,219 kb. To construct the map, probes corresponding to single-copy chromosomal sequences were used in Southern blots of digested DNA separated on pulsed-field gels, to determine how the fragments from different digests overlapped. Some of the probes represented identified gonococcal genes, whereas others were anonymous cloned fragments of strain FA1090 DNA. By using this approach, a macrorestriction map of the strain FA1090 chromosome was assembled, and the locations of various genetic markers on the map were determined. Once the map was completed, the repeated gene families encoding Opa and pilin proteins were mapped. The 11 opa loci of strain FA1090 were distributed over approximately 60% of the chromosome. The pil loci were more clustered and were located in two regions separated by approximately one-fourth of the chromosome.

Antigenic variation of gonococcal pilin involves a family of variable genes that undergo homologous recombination, resulting in transfer of variant sequences from the pilS silent gene copies into the complete pilE expression locus. Little is known about the specific recombination events that are involved in assembling new variant pilin genes in vivo. One approach to understanding pilin variation in vivo is to carry out experimental human infections with a gonococcal strain having a fully characterized repertoire of pilin genes, so that the specific recombination events occurring in vivo can be determined. To this end, the authors cloned, sequenced and mapped the pilin genes of strain FA1090 of Neisseria gonorrhoeae. This strain contains one pilE locus and 19 silent gene copies that are arranged in five pilS loci ; the pilE locus and four of the pilS loci are clustered in a 35 kb region of the chromosome. The general features of the pilin loci in FA1090 are similar to those in strain MS11, in which the mechanism of pilin variation has been extensively studied. However, none of the silent copy sequences are identical in the two strains, which emphasizes the extreme variability in this gene family among gonococci. Three male volunteers were inoculated with the same variant of strain FA1090 and developed urethritis within 2-4 d. The pilE gene sequences from a total of 23 colonies cultured from the subjects were analysed, determining which pilS silent copy donated each portion of the expressed pilE genes. There were 12 different pilin variants, one of which was the original inoculum variant, among the in vivo-expressed pilE gene sequences. The pilE of the inoculum variant was derived entirely from a single silent copy (pilS6c1). However, the pilE genes in the majority of the colonies cultured from the infected subjects were chimeras of sequence derived from two or three silent copies. Recombination to generate new pilE sequences involved exchange of single variable minicassettes, multiple minicassettes, entire silent gene copies, or (rarely) recombination within a minicassette.

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.

Class 5 outer membrane proteins of Neisseria meningitidis show both phase- and antigenic variation of expression. The proteins are encoded by a family of opa genes that share a conserved framework interspersed with three variable regions, designated the semivariable (SV) region and hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of all of the opa genes of meningococcal strain FAM18, to assess the structural and antigenic variability in the family of proteins made by one strain. Pulsed field electrophoresis and Southern blotting showed that there are four opa genes in the FAM18 chromosome, and that they are not tightly clustered. DNA sequence analysis of the four cloned genes showed a modest degree of diversity in the SV region and more extensive differences in the HV1 and HV2 regions. There were four versions of HV1 and three versions of HV2 among the four genes. Each of the FAM18 opa loci contained a gene with a unique combination of SV, HV1, and HV2 sequences. We used lambda gt11 cloning and synthetic peptides to demonstrate that HV2 sequences completely encode the epitopes for two monoclonal antibodies specific for different class 5 proteins of FAM18.

A physical map of the chromosome of N. meningitidis Z2491 (serogroup A, subgroup IV-1) has been constructed. Z2491 DNA was digested with NheI, SpeI, SgfI, PacI, BglII, or PmeI, resulting in a limited number of fragments that were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis. The estimated genome size for this strain was 2,226 kb. To construct the map, probes corresponding to single-copy genes or sequences were used on Southern blots of chromosomal DNA digested with the different mapping enzymes and subjected to CHEF electrophoresis. By determining which fragments from different digests hybridized to each specific probe, it was possible to walk back and forth between digests to form a circular macrorestriction map. The intervals between mapped restriction sites range from 10 to 143 kb in size. A total of 117 markers have been placed on the map; 75 represent identified genes, with the remaining markers defined by anonymous cloned fragments of neisserial DNA. Comparison of the arrangement of genetic loci in Z2491 with that in gonococcal strain FA1090, for which a physical map was previously constructed, revealed complex genomic rearrangements between the two strains. Although gene order is generally conserved over much of the chromosome, a region of approximately 500 kb shows translocation and/or inversion of multiple blocks of markers between the two strains. Even within the relatively conserved portions of the maps, several genetic markers are in different positions in Z2491 and FA1090.